[microwave-assisted peptide synthesis]

ABSTRACT

An instrument and process for accelerating the solid phase synthesis of peptides is disclosed. The method includes the steps of deprotecting a protected first amino acid linked to a solid phase resin by admixing the protected linked acid with a deprotecting solution in a microwave transparent vessel while irradiating the admixed acid and solution with microwaves, then activating a second amino acid by adding the second acid and an activating solution to the same vessel while irradiating the vessel with microwaves, then coupling the second amino acid to the first acid while irradiating the composition in the same vessel with microwaves, and cleaving the linked peptide from the solid phase resin by admixing the linked peptide with a cleaving composition in the same vessel while irradiating the composition with microwaves.

BACKGROUND OF INVENTION

[0001] The present invention relates to solid-phase peptide synthesis(SPPS), and in particular relates to microwave-assisted techniques forSPPS.

[0002] The early part of the twentieth century saw the birth of a novelconcept in scientific research in that synthetically produced peptidescould greatly facilitate the study of the relationship between chemicalstructure and biological activity. Until that time, the study ofstructure-activity relationships between peptides and their biologicalfunction had been carried out using purified, naturally occurringpeptides. Such early, solution-based techniques for peptide purificationwere plagued with problems, however, such as low product yield,contamination with impurities, their labor-intensive nature and theunpredictable solubility characteristics of some peptides. During thefirst half of the twentieth century some solution-based synthesistechniques were able to produce certain “difficult” peptides, but onlyby pushing known techniques to their limits. The increasing demand forhigher peptide yield and purity resulted in a breakthrough techniquefirst presented in 1963 for synthesizing peptides directly from aminoacids, now referred to as solid-phase peptide synthesis (SPPS).

[0003] The drawbacks inherent in solution-based peptide synthesis haveresulted in the near-exclusive use of SPPS for peptide synthesis. Solidphase coupling offers a greater ease of reagent separation, eliminatesthe loss of product due to conventional chemistries (evaporation,recrystalization, etc.), and allows for the forced completion of thereactions by adding excess reagents.

[0004] Peptides are defined as small proteins of two or more amino acidslinked by the carboxyl group of one to the amino group of another.Accordingly, at its basic level, peptide synthesis of whatever typecomprises the repeated steps of adding amino acid molecules to oneanother or to an existing peptide chain of acids.

[0005] The synthetic production of peptides is an immeasurably valuabletool in the field of scientific research for many reasons. For example,some antiviral vaccines that exist for influenza and the humanimmunodeficiency virus (HIV) are peptide-based. Likewise, some work hasbeen done with antibacterial peptide-based vaccines (diphtheria andcholera toxins). Synthetically altered peptides can be labeled withtracers, such as radioactive isotopes, and used to elucidate thequantity, location, and mechanism of action of the native peptide'sbiological acceptor (known as a receptor). This information can then beused to design better drugs that act through that receptor. Peptides canalso be used for antigenic purposes, such as peptide-based antibodies toidentify the protein of a newly discovered gene. Finally, some peptidesmay be causative agents of disease. For example, an error in thebiological processing of the beta-amyloid protein leads to the“tangling” of neuron fibers in the brain, forming neuritic plaques. Thepresence of these plaques is a pathologic hallmark of Alzheimer'sDisease. Synthetic production of the precursor, or parent molecule, ofbeta-amyloid facilitates the study of Alzheimer's Disease.

[0006] These are, of course, only a few of the wide variety of topicsand investigative bases that make peptide synthesis a fundamentalscientific tool.

[0007] The basic principle for SPPS is the stepwise addition of aminoacids to a growing polypeptide chain that is anchored via a linkermolecule to a solid phase particle which allows for cleavage andpurification once the coupling phase is complete. Briefly, a solid phaseresin support and a starting amino acid are attached to one another viaa linker molecule. Such resin-linker-acid matrices are commerciallyavailable (e.g., Calbiochem, a brand of EMD Biosciences, an affiliate ofMerck KGaA of Darmstadt, Germany; or ORPEGEN Pharma of Heidelberg,Germany, for example). The starting amino acid is protected by achemical group at its amino terminus, and may also have a chemicalside-chain protecting group. The protecting groups prevent undesired ordeleterious reactions from taking place at the alpha-amino group duringthe formation of a new peptide bond between the unprotected carboxylgroup of the free amino acid and the deprotected alpha-amino of thegrowing peptide chain. A series of chemical steps subsequently deprotectthe amino acid and prepare the next amino acid in the chain for couplingto the last. Stated differently, “protecting” an acid prevents undesiredside or competing reactions, and “deprotecting” an acid makes itsfunctional group(s) available for the desired reaction.

[0008] When the desired sequence of amino acids is achieved, the peptideis cleaved from the solid phase support at the linker molecule. Thistechnique consists of many repetitive steps making automation attractivewhenever possible.

[0009] Many choices exist for the various steps of SPPS, beginning withthe type of reaction. SPPS may be carried out using a continuous flowmethod or a batch flow method. Continuous flow is useful because itpermits real-time monitoring of reaction progress via aspectrophotometer. However, continuous flow has two distinctdisadvantages in that the reagents in contact with the peptide on theresin are diluted, and scale is more limited due to physical sizeconstraints of the solid phase resin. Batch flow occurs in a filterreaction vessel and is useful because reactants are accessible and canbe added manually or automatically.

[0010] Other choices exist for chemically protecting the alpha-aminoterminus. A first is known as “Boc” (N-Î±-t-butoxycarbonyl). Althoughreagents for the Boc method are relatively inexpensive, they are highlycorrosive and require expensive equipment. The preferred alternative isthe “Fmoc” (9-fluorenylmethyloxycarbonyl) protection scheme, which usesless corrosive, although more expensive, reagents.

[0011] For SPPS, solid support phases are usually polystyrenesuspensions;

[0012] more recently polymer supports such as polyamide have also beenused. Preparation of the solid phase support includes “solvating” it inan appropriate solvent (dimethyl formamide, or DMF, for example). Thesolid phase support tends to swell considerably in volume duringsolvation, which increases the surface area available to carry outpeptide synthesis. As mentioned previously, a linker molecule connectsthe amino acid chain to the solid phase resin. Linker molecules aredesigned such that eventual cleavage provides either a free acid oramide at the carboxyl terminus. Linkers are not resin-specific, andinclude peptide acids such as4-hydroxymethylphenoxyacetyl-4′-methylbenzyhydrylamine (HMP), or peptideamides such as benzhydrylamine derivatives.

[0013] Following the preparation of the solid phase support with anappropriate solvent, the next step is to deprotect the amino acid to beattached to the peptide chain. Deprotection is carried out with a mildbase treatment (picrodine or piperidine, for example) for temporaryprotective groups, while permanent side-chain protecting groups areremoved by moderate acidolysis (trifluoroacetic acid, or TFA, as anexample).

[0014] Following deprotection, the amino acid chain extension, orcoupling, is characterized by the formation of peptide bonds. Thisprocess requires activation of the C-alpha-carboxyl group, which may beaccomplished using one of five different techniques. These are, in noparticular order, in situ reagents, preformed symmetrical anhydrides,active esters, acid halides, and urethane-protected N-carboxyanhydrides.The in situ method allows concurrent activation and coupling; the mostpopular type of coupling reagent is a carbodiimide derivative, such asN,N″-dicyclohexylcarbodiimide or N,N-diisopropylcarbodiimide.

[0015] After the desired sequence has been synthesized, the peptide iscleaved from the resin. This process depends on the sensitivity of theamino acid composition of the peptide and the side-chain protectorgroups. Generally, however, cleavage is carried out in an environmentcontaining a plurality of scavenging agents to quench the reactivecarbonium ions that originate from the protective groups and linkers.One common cleaving agent is TFA.

[0016] In short summary SPPS requires the repetitive steps ofdeprotecting, activating, and coupling to add each acid, followed by thefinal step of cleavage to separate the completed peptide from theoriginal solid support.

[0017] Two distinct disadvantages exist with respect to current SPPStechnology. The first is the length of time necessary to synthesize agiven peptide. Deprotection steps can take 30 minutes or more. Couplingeach amino acid to the chain as described above requires about 45minutes, the activation steps for each acid requires 15-20 minutes, andcleavage steps require two to four hours. Thus, synthesis of a meretwelve amino acid peptide may take up to 14 hours. To address this,alternative methods of peptide synthesis and coupling have beenattempted using microwave technology. Microwave heating can beadvantageous in a large variety of chemical reactions, including organicsynthesis because microwaves tend to interact immediately and directlywith compositions or solvents. Early workers reported simple couplingsteps (but not full peptide synthesis) in a kitchen-type microwave oven.Such results are not easily reproducible, however, because of thelimitations of a domestic microwave oven as a radiation source, a lackof power control, and reproducibility problems from oven to oven. Othershave reported enhanced coupling rates using microwaves, but haveconcurrently generated high temperatures that tend to cause the solidphase support and the reaction mixtures to degenerate. Sample transferbetween steps has also presented a disadvantage.

[0018] Another problem with the current technology is aggregation of thepeptide sequence. Aggregation refers to the tendency of a growingpeptide to fold back onto itself and form a loop, attaching via hydrogenbonding. This creates obvious problems with further chain extension.Theoretically, higher temperatures can reduce hydrogen bonding and thusreduce the fold-back problem, but such high temperatures can createtheir own disadvantages because they can negatively affectheat-sensitive peptide coupling reagents. For this reason, SPPSreactions are generally carried out at room temperature, leading totheir characteristic extended reaction times.

SUMMARY OF INVENTION

[0019] In one aspect, the invention is a process for the solid phasesynthesis of peptides, which comprises the steps of: (a) deprotecting afirst amino acid linked to a solid phase resin by removing protectivefirst chemical groups; (b) activating chemical groups on a second aminoacid to prepare the second amino acid for coupling with the first aminoacid; (c) coupling the activated second amino acid to the deprotectedfirst amino acid to form a peptide from the first and second aminoacids; and (e) applying microwave energy to accelerate the deprotecting,activating, and coupling cycle.

[0020] In another aspect the invention is an apparatus for theaccelerated synthesis of peptides by the solid phase method, thatcomprises a reaction cell that is transparent to microwave radiation; apassageway for adding liquids to the reaction cell; a passageway forremoving liquids but not solids from the reaction cell; a microwavecavity for holding the cell; and a microwave source in wavecommunication with the cavity.

[0021] In yet another aspect, the invention is a vessel system for theaccelerated synthesis of peptides by the solid phase method, the vesselsystem comprising: a reaction cell that is transparent to microwaveradiation; a first passageway in fluid communication with the cell fortransferring solid phase resin between a resin source external to thecell and the cell; a second passageway in fluid communication between atleast one amino acid source and the cell for adding amino acids to thecell; a third passageway in gaseous communication with an inert gassource and with a vent for applying gas pressure to and releasing gaspressure from the cell so that the controlled flow of gases to and fromthe cell can be used to add and remove fluids and flowing solids to andfrom the cell.

[0022] In yet another aspect the invention is a process for acceleratingthe solid phase synthesis of peptides, and comprising: deprotecting aprotected first amino acid linked to a solid phase resin by admixing theprotected linked acid with a deprotecting solution in a microwavetransparent vessel while irradiating the admixed acid and solution withmicrowaves; activating a second amino acid by adding the second acid andan activating solution to the same vessel while irradiating the vesselwith microwaves; coupling the second amino acid to the first acid whileirradiating the composition in the same vessel with microwaves; andcleaving the linked peptide from the solid phase resin by admixing thelinked peptide with a cleaving composition in the same vessel whileirradiating the composition with microwaves.

BRIEF DESCRIPTION OF DRAWINGS

[0023]FIG. 1 is a schematic diagram illustrating certain aspects ofsolid phase peptide synthesis.

[0024]FIG. 2 is a perspective view of a synthesis instrument accordingto the present invention.

[0025]FIGS. 3, 4 and 5 are perspective views of a reaction vessel andadapter according to the present invention.

[0026]FIG. 6 is a flow circuit diagram illustrating aspects of thepresent invention.

[0027]FIG. 7 is a cut-away perspective view of the cavity and waveguideof the present invention.

[0028]FIG. 8 is the mass spectrum of one peptide synthesized accordingto the method of the invention.

[0029]FIG. 9 is the mass spectrum of a second peptide synthesizedaccording to the method of the invention.

DETAILED DESCRIPTION

[0030] The invention is an apparatus and method for the solid phasesynthesis of one or more peptides, specifically utilizing microwaveenergy to accelerate the method.

[0031]FIG. 1 is a schematic diagram illustrating some aspects of thesolid phase peptide synthesis process. It will be understood that FIG. 1is general in nature and is not limiting of the invention. FIG. 1illustrates a first amino acid 10 that includes an N-alpha protectivegroup 11 and a side chain protective group 12 attached to it. A linkingmolecule 13 is attached to a resin support 14. In a first stepdesignated by the arrow 15, the first acid and its protective groups 11and 12 are attached to the linker 13 and the resin support 14. In asecond step indicated by the arrow 17, the N-alpha protective group isremoved (“deprotected”) to produce the structure in which the first acid10 and its side chain-protecting group 12 are linked to the support 14through the linker molecule 13. In the next step, indicated by the arrow21, the first amino acid 10 is coupled to a second amino acid designatedat 20, which similarly has an N-alpha protective group 11 and anactivation group 22 attached to it to encourage the coupling. Followingthe coupling step 21, the resulting structure includes the first acid 10and the second acid 20 connected to one another and still including theN-alpha protective group 11 attached to the second acid 20 and the sidechain protective group 12 attached to the first acid 10 with theconnected acids being in turn linked to the support 14 through thelinking molecule 13. Additional acids, represented by the brokenrectangle 25 are added in the same manner (arrow 21″) to lengthen thepeptide chain as desired.

[0032] In the final step, the connected acids 10, 20 and 25 are cleaved,represented by the arrow 23, from the protective groups and the supportto result in the desired peptide separate from the resin support 14. Thecoupling steps can, as indicated a number of times elsewhere herein, berepeated as many times as desired to produce a resulting peptide.

[0033]FIG. 2 illustrates one commercial embodiment of the presentinvention broadly designated at 30. FIG. 2 illustrates some of the broadstructural aspects of the invention, the details of which will beexplained with respect to FIGS. 3 and 6.

[0034] First, FIG. 2 illustrates the microwave portion of the device 31.The portion of the instrument that applies microwave irradiation to thevessel is preferably a single-mode cavity instrument that can becontrolled to apply suitable amounts of power to the sample sizes andmaterials used in the method of the invention. In the preferredembodiment of the invention, the microwave portion of the instrument hasthe design and operation that is set forth in a number of co-pending andcommonly assigned U.S. patent applications. These include publishedapplications Nos. (U.S.) 20030089706 and 20020117498, along withyet-to-be-published application Ser. Nos. 10/126,838 filed Apr. 19,2002; Ser. No. 10/064,261 filed Jun. 26, 2002; and Ser. No. 10/064,623filed Jul. 31, 2002. The disclosures of all of these references areincorporated entirely herein by reference. Commercial versions of suchsingle-mode microwave instruments are available from the assignee of thepresent invention, CEM Corporation, of Matthews, N.C., under theDISCOVERYâ

, VOYAGERâ

, and EXPLORERâ

trade names.

[0035] With those considerations in mind, FIG. 2 illustrates thelocation of the cavity 32, the housing 33, and an appropriate display34, for providing instructions or information during operation. Aplurality of amino acid source containers or bottles are eachrespectively indicated at 35. The respective resin containers areillustrated at 36, and the product peptide containers are designated at37. A series of fluid passageways are illustrated by the portions oftubing broadly designated at 40 and will be discussed in more detailwith respect to FIG. 6. Similarly, the instrument 30 includes an upperhousing portion 41, which includes an appropriate manifold, forphysically transporting the fluids and resins in the manner describedherein. Although the manifold is not illustrated, it can comprise anyseries of passageways and valves that serve to direct the fluids in themanner described herein and particularly described with respect to thecircuit diagram of FIG. 6.

[0036] Thus, in the embodiment illustrated in FIG. 2, up to 20 differentamino acids can be incorporated in the respective containers 35, and upto 12 different peptides can be produced and placed in the respectivecontainers 37 in automated fashion. It will be understood that these arecommercial embodiment numbers, however, and that the invention isneither limited to this number nor does it need to have as many sourcesor product containers as are illustrated.

[0037]FIG. 2 also illustrates a complimentary series of passagewaysshown as the tubing broadly designated at 42 that are immediatelyconnected to the reaction vessel adapter 43, which is partiallyillustrated in FIG. 2, but is described in more detail with respect toFIGS. 3, 4, and 5.

[0038]FIG. 3 is a partial perspective view of the reaction vessel 45 andthe vessel adapter 43, portions of which were also illustrated in FIG.2. The reaction vessel 45 is preferably pear-shaped and formed of amaterial that is transparent to microwave radiation. Preferred materialsinclude, but are not limited to, glass, Teflon, and polypropylene. Afirst passageway, shown as the tubing 46, is in fluid communication withthe reaction vessel (or “cell,” the terms are used interchangeableherein) 45 for transferring solid phase resin between a resin sourceexternal to the cell 45 and the cell 45. A second passageway 47 is influid communication between at least one amino acid source (FIG. 6) andthe cell 45 for adding amino acids to the cell 45. A third passageway 50is in gas communication with an inert gas source (FIG. 6) and with avent (FIG. 6) for applying and releasing gas pressure to and from thecell 45, so that the controlled flow of gas in the manifold and to andfrom the cell 45 can be used to add and remove fluids and flowing solidsto and from the cell 45.

[0039]FIG. 3 also illustrates that the second passageway 47 alsoincludes a filter, shown as the frit 51, for preventing solid-phaseresin from entering the second passageway 47 from the cell 45.

[0040] In preferred embodiments, the invention further comprises afourth passageway 52, in fluid communication between an external solventsource (FIG. 6) and the cell 45 for flushing the cell 45 with solvent.As illustrated in FIG. 3, the fourth passageway 52 includes a spray head53 or equivalent structure for adding the solvent to the cell 45.

[0041] The adapter 43 is formed of a microwave transparent andchemically inert material, which is preferably formed of a polymer, suchas a fluorinated polymer (e.g., PTFE) or an appropriate grade ofpolypropylene. The adapter 43 is preferably a solid cylinder with thepassageways 46, 47, 50, and 52 drilled or bored there through. Thepassageways 46, 47, 50, 52 can simply comprise the bore holes throughthe adapter 43, but preferably may also include tubing, which again isformed of a microwave transparent, chemically inert material such asPTFE, PTFE variations, or polypropylene. The tubing is preferably{fraction (1/8)} inch outside diameter and {fraction (1/16)} inch insidediameter.

[0042] Although not illustrated in FIG. 3 (to reduce the complexity ofthe drawing), the vessel neck 54 preferably is externally threaded andengages an internally threaded bore hole 55 in lower portions of theadapter 43. The threaded engagement between the vessel 45 and theadapter 43 permits secure engagement between these two items, and alsopermits the vessel 45 to be easily engaged and disengaged to and fromthe adapter 43. In particular, differently sized vessels or vesselsformed of different materials can be substituted and still fit theadapter 43, provided the necks are of the same size and threading.

[0043] As some final details, FIG. 3 also includes threaded fittings 56,57, 60, and 62 to the respective first, second, third and fourthpassageways 46, 47, 50 and 52. These permit the entire adapter 43 andvessel 45 to be easily connected to and removed from the remainder ofthe instrument 30.

[0044]FIGS. 4 and 5 are respective assembled and exploded perspectivedrawings of the adapter of FIG. 3, and thus illustrate the sameelements. Both figures include the adapter 43 and the cell 45. Thethreaded fittings 57, 60, 56, and 62 are visible in FIG. 5, with 57, 60and 56 also visible in 54. The exploded view of FIG. 5 also illustratesportions of the first and second passageways, 46, 47, as well as thethreaded vessel neck 54 and the board opening 55 in the lower portionsof the adapter 43.

[0045]FIG. 6 is a flow circuit diagram for the present invention.Wherever possible, the elements illustrated in FIG. 6 will carry thesame reference numerals as in the other drawings. Because most of theelements symbolized in FIG. 6 are commonly available and wellunderstood, they will not be described in particular detail, as those ofskill in this art can practice the invention based on FIG. 6 withoutundue experimentation.

[0046] Accordingly, FIG. 6 illustrates a vessel system for theaccelerated synthesis of peptides by the solid-phase method. The vesselsystem comprises the reaction cell (or vessel) 45, which is indicated inFIG. 6 schematically as a square. Otherwise, the reaction cell 45 hasall of the characteristics already described and which will not berepeated with respect to Figure The first passageway 46 is in fluidcommunication with the cell 45 for transferring solid phase resinbetween an external resin source 36 and the cell 45. Three resinsources, 36 (A, B and C) are illustrated in FIG. 6 and correspond to theresin sources 36 illustrated in FIG. 2. As set forth with respect toFIG. 2, the number of resin sources is elective rather than mandatorywith 12 being shown in the embodiment of FIG. 2, and three illustratedin FIG. 6 for purposes of simplicity and schematic understanding. Eachof the resin sources 36 is in communication with a respective three-wayvalve 64, A, B and C, and in turn, to an appropriate resin line 65, A, Band C and then another three-way valve 66 adjacent to cell 45 fordelivering resin through the first passageway 46 into the cell 45. Thethree-way valve 66 is immediately in communication with anotherthree-way valve 67, the purpose of which will be described shortly.

[0047]FIG. 6 also shows the second passageway 47, which is incommunication with at least one of the amino acid sources 35, which areillustrated again as rectangles in the upper portions of FIG. 6. Theschematically illustrated amino acid sources or containers 35 correspondto the containers 35 illustrated in FIG. 2.

[0048] The third passageway 50 is in gas communication with an inert gassource 70 and with a vent 71 for applying gas pressure to and releasinggas pressure from the cell 45, so that the controlled flow of gasses toand from the cell 45 can be used to add and remove fluids and flowingsolids to and from the cell. The third passageway 50 accomplishes thisin conjunction with at least one valve 72 which, depending upon itsorientation, permits the third passageway 50 to communicate with eitherthe gas source 70 or the vent 71. The gas source can be any gas that canappropriately be pressurized and that does not otherwise interfere withthe chemistry of the peptide synthesis or the elements of the instrumentitself. Thus, a number of inert gases are suitable, with pressurizednitrogen being typically favored for reasons of wide availability, lowercost, ease of use, and lack of toxicity. FIG. 6 illustrates that thenitrogen supply 70 is controlled through a two-way valve 72 and anappropriate regulator 73, which also may include a filter. In theorientation of FIG. 6, the gas line from the two-way valve 72 to thevent 71 is labeled at 74, and the passageway from the valve 72 to theregulator 73 is designated at 75.

[0049]FIG. 6 also illustrates the filter 51 in the second passageway 47for preventing the solid phase resin from entering the second passagewayfrom the cell 45.

[0050]FIG. 6 also illustrates the fourth passageway 52 along with thespray head 53. As described with respect to FIG. 3, the fourthpassageway 52 is in fluid communication with one or more externalsolvent sources three of which are illustrated at 76, A, B and C. Twoother external solvent sources 77 and 80 are separately labeled becauseof their optionally different fluid paths.

[0051]FIG. 6 also illustrates the manner in which the pressurized gasfrom the source 70 can be used to both deliver compositions to, and thenremove them from, the reaction cell 45 as desired whether they bepeptides, solvent, wastes, or resin. Thus, in one aspect of suchdelivery, FIG. 6 illustrates a gas passage 81 that communicates withseveral items. First, the gas passageway 81 communicates with a seriesof two-way valves designated at 82A, B, C and D that each provide a gaspassage when the respective valve is open to its corresponding aminoacid container 35. Pressurized gas entering a container 35 pushes theacid through the respective delivery lines 83A, B, C or D, which in turncommunicates with a respective acid valves 84 A, B, C and D and thenwith the second passageway 47 and its respective two-way valve 85 andthree-way valve 86. To illustrate, when valves 82A and 84A are open, andthe remaining valves 82B, C and D are closed, gas from the source 70 canbe directed through the gas passage 81, through valve 82A, into aminoacid bottle 35A, from the bottle 35A through the valves 84A, 85 and 86,and then into the cell 45.

[0052] The respective valves are automated in order to provide the cellwith the desired composition (e.g. resin, solvent, acid) at theappropriate point in the synthesis, as well as to remove compositionsfrom the cell (peptides, waste) at other appropriate points. Therequired programming and processor capacity is well within thecapability of a personal computer-type processor (eg. PENTIUM III^(Å®)),and the use of automated controls and sequences is generally wellunderstood in this and related arts, e.g. Dorf, The ElectricalEngineering Handbook, 2d Ed. (CRC Press 1997).

[0053] It should be understood that while many amino acids exist, thetwenty source containers of this apparatus are intended, but not limitedto, contain the twenty “common” amino acids for synthesizing proteinsthat are well known to those skilled in this art. These commerciallyavailable common amino acids can be purchased in chemically “protected”form (also from Sigma-Aldrich) to prevent unwanted and/or deleteriousreactions from occurring.

[0054] Solvent can be delivered to the cell in an analogous manner. Thesolvents communicate with the gas passage 81 through the valves 87A, Band C and 90 and 91. This places the gas in direct communication withthe external solvent tanks 76A, B and C and 77 and 80. External solventtanks 76A, B and C are further in communication with respective two-wayvalves 92A, B and C and respective three-way valves 93 and 94. These alllead, when the valves are appropriately oriented, to the secondpassageway 47 for delivering solvent to the reaction vessel 45 using gaspressure in the same manner that the acids are delivered. A TFA solventis used in external reservoirs 76C and thus can be directed throughalternative lines for optional isolation.

[0055]FIG. 6 also indicates that the gas source 70 can be used to driveitems from the cell 45 directly by closing all of the valves to theamino acids and the external solvent reservoirs, and then directing thegas through the regulator and filter 92 and its associated passageway 93directly to valves 67 and 66 and then into the first passageway 46 andthe cell 45.

[0056] Alternatively, the first passageway 46 can be used to empty thecell 45. In this aspect, valve 72 is set to direct gas from the source70 and through the passage 75 to the valve 72 and through the thirdpassageway 50 and into the cell. The gas pressure then directs fluids inthe cell 45 through either second passageway 47 or first passageway 46depending upon the orientation of the valves 86, 66 and 67. FIG. 6 alsoillustrates an additional three-way valve 95 that can direct product tothe product containers 37A, B and C, which correspond to the productcontainers 37 illustrated in FIG. 2. An appropriate set of productvalves 96A, B and C can be opened or closed as desired to direct thedesired peptide product to the desired product container 37A, B or C.

[0057] Alternatively, depending upon the orientation of valves 86, 66,67 and 95, and together with additional two-way valve 100 and three-wayvalve 101 adjacent to waste containers 102A and 102B, materials can bedirected from the cell 45 to either of the waste containers 102A and B.

[0058] The pressurized gas from the source 70 can also be used todeliver resin. In this aspect, the pressurized gas is directed throughthe gas passage 81 and through the three-way valves 103 and 104. Withrespect to delivery of resin, however, when both of the valves 103 and104 are open to the resin containers, they direct the pressurized gas tothree respective valves 105A, B and C which in turn are in communicationwith the resin containers 36 and the exit valves 64A, 64B and 64C whichthen use the gas pressure delivered to force the resin through the resinline 65 and eventually to the first passage 46 for delivery into thereaction vessel 45.

[0059] The resin sources may contain variable amounts and kinds ofresins, including, but not limited to, Wang resins, Trityl resins, andRink acid labile resins; the resins are commercially available fromvendors such as Sigma-Aldrich Corp., Saint Louis, Mo. 63101.

[0060] Solvent can be directed to the resin containers 36A, B, C, fromthe external reservoirs 77, 80 using the valves 103, 104 between thesolvent reservoirs and the resin containers.

[0061]FIG. 6A is a more detailed illustration of the valving systemadjacent the reaction vessel 45. In particular, FIG. 6A shows a seriesof liquid sensors 106, 107 and 110 in conjunction with a series ofthree-way valves 111, 112, 113, 114 and 115. The operation of the valvesin accordance with the sensors permits a metered amount of liquid to beadded to the reaction vessel 45 as may be desired or necessary. Forexample, with the valves 111, 113 and 114 shown in the orientation ofFIG. 6A, fluid can flow directly from valve 86 all the way to thoseportions of second passageway 47 that extend immediately into thereaction vessel 45. Alternatively, if valve 111 is open towards valve112, liquid will flow through valves 111 and 112 until it reaches theliquid sensors 107 and 110. The liquid sensors inform the system when aproper or desired amount of liquid is included, which can then bedelivered by changing the operation of valve 112 to deliver to the valve113, and then to the valve 114, and then to the second passageway 47 andfinally into the cell 45 as desired.

[0062] Thus, in overall fashion, FIG. 6 illustrates the delivery ofprecursor compositions (amino acids, solvents, resin, deprotectants,activators) from their respective sources to the single reaction celland the further delivery of products and by-products (peptides, waste,cleaved resin) from the cell to their respective destinations. It willbe understood that the particular flow paths and valve locationsillustrate, rather than limit, the present invention.

[0063] As noted earlier, the microwave instrument portions of thesynthesis instrument can essentially be the same as those set forth in anumber of commonly assigned and co U.S. applications. Accordingly, FIG.7 is included to highlight certain aspects of the microwave portion ofthe instrument without overly burdening the specification herein. Inparticular, FIG. 7 is essentially the same as FIG. 1 in previouslyincorporated application Ser. No. ______ filed Jun. 26, 2002. FIG. 7illustrates a microwave cavity 117 shown in cutaway fashion for clarity.The cavity is attached to a wave guide 120, which is in microwavecommunication with an appropriate source (not shown). Microwave sourcesare widely available and well understood by those of ordinary skill inthis art, and include magnetrons, klystrons, and solid state diodes.FIG. 7 illustrates a test tube-shaped cell 121 in the cavity 117 andsuch can be used if desired for the reactions of the present invention,although the pear-shaped vessel 45 is generally preferred.

[0064] In order to carry out the simultaneous cooling, the instrumentincludes a cooling gas source (not shown) which delivers the cooling gasto the inlet fitting 122 on the flow valve 123 (typically a solenoid).During active cooling, the solenoid 123, which is typically softwarecontrolled, directs cooling gas through the tubing 124 and to thecooling nozzle 125, which directs the cooling gas on to the reactionvessel 121.

[0065] It should be pointed out, however, that other cooling mechanismsmay be adapted to this method, such as a stream of refrigerated air or aliquid cooling mechanism that circulates refrigerated liquid around thereaction cell in a manner that would not interfere with the transfer ofmicrowave energy.

[0066]FIG. 7 also illustrates a cylindrical opening 126, which istypically used to permit temperature observation of the reaction vial121. Such temperature observation can be carried out with anyappropriate device, which can normally include a fiber optic device ofthe type that can measure the temperature of an object by reading theinfrared radiation produced by the object. Such devices are wellunderstood in the art, and will not be discussed in further detailherein, some aspects having already been discussed in the incorporatedreferences.

[0067] In preferred embodiments, the microwave source is capable of, butnot limited to, “spiking” microwave energy. In other words, themicrowave source is capable of generating high power for a short lengthof time as opposed, but not limited to, low power for a longer period oftime. This feature aids in preventing the undesirable effect ofoverheating the contents of the reaction vessel and appears to increasethe rate of reaction as well.

[0068] The apparatus optionally includes an infrared photosensor formeasuring temperature. The infrared sensor does not contact the reactioncell contents, yet still accurately measures the average temperature ofthe reaction cell contents and not merely the air temperaturesurrounding the contents. Infrared temperature analysis is moreaccurate, non-intrusive, and allows for a more simplified apparatusdesign compared to a probe or the like, which measures only a localizedarea and would require physical contact of the contents.

[0069] The second passageway is further characterized by a filter whichprevents the passage of resin. Additionally, the first and secondpassageways are in fluid communication with each other with respect tothe movement of liquid solvents and flowing solids; herein the term“flowing solids” refers to resin, with or without amino acids orpeptides attached, and suspended in an appropriate solvent.

[0070] In another aspect, the invention is a method for the solid phasesynthesis of one or more peptides that incorporates the use of microwaveenergy. Microwave energy applied to the contents of the reaction cellduring the deprotecting, activating, coupling, and cleaving stepsgreatly decreases the length of time necessary to complete thesereactions. The method for applying microwave energy may be moderated bythe microwave source in such a way as to provide the fastest reactiontime while accumulating the least amount of heat, thus more microwaveenergy may be applied and heat-associated degradation of the reactioncell contents does not occur. This method includes, but is not limitedto, spiking the microwave energy in large amounts for short lengths oftime.

[0071] The method optionally includes the synthesis of a completepeptide of two or more amino acids in a single reaction vessel, and mayinclude the coupling of one or more amino acids to one or more aminoacids that are attached to the solid phase resin.

[0072] The method includes cooling the reaction cell, and thus itscontents, during and between applications of microwave energy up to andincluding the final cleaving step. The cooling mechanism of the methodoperates during amino acid extension cycles, the term “cycle” usedherein to refer to the deprotection, activation, and coupling necessaryto link one amino acid to another. The cooling system can also operateduring and between applications of microwave energy in a given cycle tokeep the bulk temperature of the reaction cell contents down. Thecooling system can also operate when the complete peptide is cleavedfrom the resin.

[0073] Alternatively, it has also been discovered that controlling thepower, rather than strictly controlling the temperature, can alsoprovide a desired control over the progress of a reaction. As notedelsewhere herein, the use of a variable or switching power supply canhelp serve this purpose, an example of which is given in commonlyassigned U.S. Pat. No. 6,288,379; the contents of which are incorporatedentirely herein by reference.

[0074] The method includes agitating the contents of the reaction cellwith nitrogen gas in order to promote maximal exposure of the resin andany attached amino acids or peptides to solvents and free amino acids.

[0075] In a preferred embodiment, the method comprises transferring afirst common amino acid linked to a resin of choice, both suspended inan appropriate solvent, to the reaction cell via pressurized nitrogengas. A deprotection solution is then pumped into the reaction cell. Thisprocess is accelerated by the application of microwave energy, and theheat generated by the microwave energy is minimized by a coolingmechanism. Multiple deprotection steps may be executed. The deprotectionsolution is then withdrawn from the reaction cell, leaving thedeprotected, common amino acid linked to the resin. After several (threeto five) resin washes of approximately one resin volume each using anappropriate solvent and removing the wash solvent, the next “free”common amino acid or acids (dissolved in solution) is added to thereaction cell along with an activating solution. The activation of thefree amino acid is accelerated by the application of microwave energy,and the reaction cell temperature is controlled by a cooling mechanismas described above. The method further comprises coupling the free aminoacid or acids to the deprotected, linked amino acid, forming a peptide,using microwave energy to accelerate the method. As above, heatgenerated by the microwave energy is minimized by a cooling mechanism.The coupling step is further preferred to include nitrogen agitation ofthe reaction cell contents. Completion of this step represents one cycleof one or more amino acid addition. Following the coupling step, theactivation solution is withdrawn and the resin is washed as above. Thecycle is repeated until the desired peptide sequence is synthesized.Upon completion of peptide synthesis, a further deprotection step may becarried out to remove protective chemical groups attached to the sidechains of the amino acids. This deprotection step is carried out asdescribed above. The resin containing the attached, completed peptide isthen washed as above with a secondary solvent to prepare the peptide forcleavage from the resin. Following the removal of the secondary solvent,cleaving solution is added to the reaction cell and cleaving isaccelerated by the application of microwave energy, and the heatgenerated by the microwave energy is minimized by a cooling mechanism.Upon completion of cleaving, the peptide product is transferred to aproduct tube. Optionally, the peptide may be “capped” at any pointduring the synthesis process. Capping is useful to terminateincompletely coupled peptides, assist in proper folding of the peptidesequence, and to provide a chemical identification tag specific to agiven peptide. However, these modifications decrease the solubility ofsynthetic peptides and thus must be carefully considered. Capping iscarried out for example, but not limited to, using acetic anhydride orfluorous capping in solid phase synthesis, or by attaching any of alarge variety of chemical groups such as biotin to either theN-terminal, C-terminal or side chain of a peptide.

[0076] In another embodiment, the invention comprises de-protectingfirst amino acid linked to a solid phase resin by removing protectivefirst chemical groups, activating chemical groups on a second amino acidto prepare the second amino acid for coupling with the first amino acid,coupling the activated the second amino acid to the de-protected firstamino acid to form a peptide from the first and second amino acids,cleaving the peptide from the solid phase resin, applying microwaveenergy to accelerate the de-protected, activating and coupling cycle,and applying microwave energy to accelerate the cleaving step.

[0077] It is, of course, the usual procedure to add a number of aminoacids to one another to form a peptide sequence. Accordingly, the methodcan, and usually, comprises repeating the de-protecting, activating andcoupling cycle to add third and successive acids to form a peptide of adesired sequence.

[0078] In that regard, it will be understood that as used herein, termssuch as “first,” “second,” or “third” are used in a relative rather thanabsolute sense.

[0079] In a particularly preferred embodiment, the method comprisessuccessively de-protecting, activating and coupling a plurality of aminoacids into a peptide in a single vessel without removing the peptidefrom the solid phase resin between the cycles. This, and additionalaspects, of the invention will be understood with regard to thediscussion of the figures.

[0080] In another embodiment, the method comprises proactively coolingthe vessel and its contents during the application of microwave energyto thereby prevent undesired degradation of the peptide or acids bylimiting heat accumulation that would otherwise result from theapplication of the microwave energy.

[0081] As is typical in peptide synthesis, the de-protecting stepcomprises de-protecting the alpha-amino group of the amino acid, but canalso comprise de-protecting side chains on the amino acids of thepeptide, both under the microwave and radiation. Similarly, theactivating step typically comprises activating the alpha-carboxyl groupof the second amino acids.

[0082] Because the amino acids and peptides are sensitive to excessiveheat, and in addition to the proactive cooling step just described, thestep of applying the microwave energy can comprise “spiking” theapplication of microwave energy to relatively short-time intervals tothereby prevent undesired degradation of the peptidal acids by limitingheat accumulation that could be encouraged by the continuous applicationof the microwave energy. As used herein, the term “spiking” refers tothe limitation of the application of microwave energy to the relativetime intervals. Alternatively, the microwave power can be supplied froma switching power supply as set forth in commonly assigned U.S. Pat. No.6,288,379, the contents of which are incorporated entirely herein byreference.

[0083] In other embodiments, the peptide synthesis process can compriseactivating and coupling in situ using a carbodiimide type coupling freeagent.

[0084] In another aspect, the invention is a process for acceleratingthe solid phase synthesis of peptides. In this aspect, the methodcomprises deprotecting a protected first amino acid linked to a solidphase resin by admixing the protective linked acid with a deprotectingsolution in a microwave, transparent vessel while irradiating theadmixed acid and solution with microwaves, and while cooling theadmixture (or alternatively controlling the applied power, or both) toprevent heat accumulation from the microwave energy from degrading thesolid phase support or any of the admixed compositions. In particular,the method comprises deprotecting the alpha-amino group of the aminoacid, and most typically with a composition suitable for removingprotective chemicals selected from the group consisting of fmoc and boc.As is known to those familiar with this chemistry, the deprotecting stepcan also comprise deprotecting the side chain of the amino acid thosecircumstances, the deprotecting step comprises using a compositionsuitable for removing tside chain protecting groups.

[0085] Following the deprotecting step, the method comprises activatinga second amino acid by adding this second amino acid and an activatingsolution to the same vessel while irradiating the vessel with microwavesand while simultaneously cooling the vessel to prevent heat accumulationfrom the microwave energy from degrading the solid face support or anyof the admixed compositions.

[0086] The method next comprises coupling the second amino acid to thefirst acid while irradiating the composition in the same vessel withmicrowaves, and while cooling the admixture to prevent heat accumulationfrom the microwave energy from degrading the solid phase support or anyof the admixed compositions.

[0087] Finally, the method comprises the step of cleaving the linkedpeptide from the solid phase resin by admixing the linked peptide with acleaving composition in the same vessel while irradiating thecomposition with microwaves, and while cooling the vessel to preventheat accumulation from the microwave energy from degrading the solidphase support or the peptide.

[0088] The activating step can also comprise activating and coupling thesecond amino acid using an in situ activation method and compositionsuch as phosphorium or uranium activators, HATU, HBTU, PyBOP, PyAOP, andHOBT.

[0089] Once again, because the synthesis of peptides almost alwaysincludes the addition of three or more acids into the chain, the methodcan comprise cyclically repeating the steps of deprotecting, activatingand coupling for three or more amino acids in succession to therebysynthesize a desired peptide.

[0090] In a particular embodiment of the invention, the successive stepsof deprotecting, activating, coupling and pleading are carried out inthe single reaction vessel without removing the peptide from the solidphase resin or from the vessel between cycles.

[0091] The method can further comprise agitating the admixture,preferably with nitrogen gas during one or more of the deprotecting,activating, coupling and pleading steps. Any gas can be used for theagitation, provided it does not otherwise interfere with the synthesischemistry, the peptides or the amino acids, but nitrogen is typicallypreferred for this purpose because of its wide availability, low costand chemical inertness with respect to the particular reactions.

[0092] Experimental:

[0093] Peptide: Asn-Gly-VaIMW=288Scale=0.10 mmolResin used=Fmoc-Val-WangResinResin substitution=0.27×10⁻³ moles/gram resinMicrowave Protocol:For all reactions in this peptide the microwave power was initially setat 50 W then regulated to maintain the temperature below 60Â° C.

[0094] Deprotection: A 20% Piperidine in DMF solution was used fordeprotection. The reaction was performed for 30 seconds in microwave,and then repeated with new deprotection solution for 1:00 minute inmicrowave.

[0095] Coupling: Activation was performed with 0.40 mmol HCTU, 0.80 mmolDIPEA, and 0.40 mmol of each Fmoc-amino acid for each coupling in thesynthesis. Approximately 10 mL of DMF was used to dissolve the mixture.The reaction was performed for 5:00 min. in the microwave.

[0096] Washing: The vessel was filled with approximately 10 mL of DMFand rinsed 5 times after each deprotection and coupling step.

[0097] Cleavage: Cleavage was performed with 95% TFA and 5% H₂O for90:00 min.

[0098] Peptide was precipitated in 50 mL of cold ethyl ether overnight.Product was collected and dried. Mass Spectrum was obtained of crudeproduct from electrospray ionization mass spectrometry using aThermoFinnigan Advantage LC/MS.

[0099] Results: The Electrospray Ionization Mass Spectrum (FIG. 8)showed a single peak at 289.1 corresponding to the MW of Asn-Gly-Val. Noother peaks corresponding to incomplete couplings were observed.

[0100] Peptide: Gly-Asn-Ile-Tyr-Asp-Ile-Ala-Ala-Gln-ValMW=1062Scale=0.25 mmolResin used: Fmoc-Val-Wang ResinResinsubstitution=0.27×10⁻³ moles/gram resinMicrowave Protocol: This peptidewas synthesized with a power time control method.

[0101] Deprotection: A 20% Piperidine in DMF solution was used fordeprotection. The deprotection was performed with 25 W of microwavepower for 30 seconds, and then repeated with new deprotection solutionfor 1:00 min. in the microwave.

[0102] Coupling: Activation was performed with 0.9/1.0 mmol of HBTU/HOBtrespectively, 2 mmol of DIPEA, and 1.0 mmol of Fmoc-amino acid for eachcoupling in the synthesis. Approximately 15 mL of DMF was used todissolve the mixture. The coupling reaction was done in 5:00 min. in themicrowave with power alternating between on for 15 seconds and off for45 seconds. The first cycle of power was 25 W, and the remaining fourwere each 20 W.

[0103] Washing: The vessel was filled with approximately 15 mL of DMFand rinsed 5 times after each deprotection and coupling step.

[0104] Cleavage: Cleavage was performed with 95% TFA, 2.5% H₂O, and 2.5%TIS.

[0105] Peptide was precipitated in 100 mL of cold ethyl ether overnight.Product was collected and dried. Mass Spectrum was obtained of productfrom electrospray ionization mass spectrometry using a ThermoFinniganAdvantage LC/MS.

[0106] Results: The Electrospray ionization mass spectrum (FIG. 9) showsa peak at 1063.3 that corresponds to the desired peptide mass. No peakswere detected for incomplete couplings. A second peak was obtained at1176.5 that corresponds to the desired peptide with an extra Ile aminoacid. This corresponds to incomplete removal of the deprotectionsolution before one of the Ile coupling reactions and allowing two Ileamino acids to be added to the peptide.

[0107] In the drawings and specification there have been disclosedtypical embodiments of the invention. The use of specific terms isemployed in a descriptive sense only, and these terms are not meant tolimit the scope of the invention being set forth in the followingclaims.

1. A process for the solid phase synthesis of peptides, which comprises:(a) deprotecting a first amino acid linked to a solid phase resin byremoving protective first chemical groups; (b) activating chemicalgroups on a second amino acid to prepare the second amino acid forcoupling with the first amino acid; (c) coupling the activated secondamino acid to the deprotected first amino acid to form a peptide fromthe first and second amino acids; and (e) applying microwave energy toaccelerate the deprotecting, activating, and coupling cycle.
 2. Aprocess according to claim 1 comprising cleaving the peptide from thesolid phase resin while applying microwave energy to accelerate thecleaving step.
 3. A peptide synthesis process according to claim 1,comprising repeating the deprotecting, activating, and coupling cycle toadd third and successive acids to form a peptide of a desired sequence.4. A peptide synthesis process according to claim 1, comprisingsuccessively deprotecting, activating, and coupling a plurality of aminoacids into a peptide in a single vessel without removing thepeptide-linked resin from the vessel between cycles.
 5. A peptidesynthesis process according to claim 1, comprising proactively coolingthe vessel and its contents during the application of microwave energyto thereby prevent undesired degradation of the peptide or acids bylimiting heat accumulation that would otherwise result from theapplication of the microwave energy.
 6. A peptide synthesis processaccording to claim 1, wherein the deprotecting step comprisesdeprotecting the alpha-amino group of the amino acid.
 7. A peptidesynthesis process according to claim 1, further comprising deprotectingside chains on the amino acids of the peptide under microwaveirradiation.
 8. A peptide synthesis process according to claim 1,wherein the activating step comprises activating the alpha-carboxylgroup of the second amino acid.
 9. A peptide synthesis process accordingto claim 1, wherein the step of applying the microwave energy compriseslimiting the application of microwave energy to relatively short timeintervals to thereby prevent undesired degradation of the peptide oracids by limiting heat accumulation that could be encouraged by thecontinuous application of the microwave energy.
 10. A peptide synthesisprocess according to claim 1 further comprising activating and couplingusing an in situ method and a composition selected from the groupconsisting of phosphorium activators, uronium activators, HATU, HBTU,PyBOP, PyAOP, and HOBT.
 11. A peptide synthesis process according toclaim 1 comprising monitoring the temperature of the vessel andmoderating the applied power based upon the monitored temperature.
 12. Apeptide synthesis process according to claim 1 comprising moderating theapplied power based on the status of the reaction.
 13. An apparatus forthe accelerated synthesis of peptides by the solid phase method, saidvessel system comprising: a reaction cell that is transparent tomicrowave radiation; a passageway for adding liquids to said reactioncell; a passageway for removing liquids but not solids from saidreaction cell; a microwave cavity for holding said cell; and a microwavesource in wave communication with said cavity.
 14. A vessel system forthe accelerated synthesis of peptides by the solid phase method, saidvessel system comprising: a reaction cell that is transparent tomicrowave radiation; a first passageway in fluid communication with saidcell for transferring solid phase resin between a resin source externalto said cell and said cell; a second passageway in fluid communicationbetween at least one amino acid source and said cell for adding aminoacids to said cell; a third passageway in gaseous communication with aninert gas source and with a vent for applying gas pressure to andreleasing gas pressure from said cell so that the controlled flow ofgases to and from said cell can be used to add and remove fluids andflowing solids to and from said cell.
 15. A peptide synthesis vesselsystem according to claim 14 comprising a processor and control systemfor controlling said passageways to sequentially add acids to the vesseland peptide and to transfer completed peptides from said vessel to apeptide reservoir.
 16. A peptide synthesis vessel system according toclaim 15 comprising means for rinsing said vessel with solvent from anexternal solvent source and thereafter sequentially adding acids to saidvessel to form a subsequent peptide in said same vessel.
 17. A peptidesynthesis vessel system according to claim 14, further comprising afilter in said second passageway for preventing solid phase resin fromentering said second passageway from said cell.
 18. A peptide synthesisvessel system according to claim 14, further comprising a fourthpassageway in fluid communication between an external solvent source andsaid cell for flushing said cell with solvent.
 19. A peptide synthesisvessel system according to claim 18, wherein said fourth passagewayterminates within said cell with a spray head mechanism.
 20. A peptidesynthesis vessel system according to claim 14, wherein said inert gassource is selected from the group consisting of pressurized nitrogen gasand pressurized argon gas.
 21. A peptide synthesis vessel systemaccording to claim 20, comprising a regulator for controlling thepressurized gas.
 22. A peptide synthesis vessel system according toclaim 14, comprising a valve system for controlling fluid communicationin said first passageway.
 23. A peptide synthesis vessel systemaccording to claim 14, comprising a valve system for controlling fluidcommunication in said second passageway.
 24. A peptide synthesis vesselsystem according to claim 14, comprising a valve system for controllinggaseous communication in said third passageway.
 25. A peptide synthesisvessel system according to claim 18, comprising a valve system forcontrolling fluid communication in said fourth passageway.
 26. A peptidesynthesis vessel system according to claim 14, wherein said first andsaid second passageways are in further fluid communication withrespective external solvent sources.
 27. A peptide synthesis vesselsystem according to claim 18, wherein said fourth passageway is infurther fluid communication with an external solvent source.
 28. Apeptide synthesis vessel system according to claim 14, comprising aresin reservoir in fluid communication with said first passageway fordepositing solid phase resin in said cell.
 29. A peptide synthesisvessel system according to claim 28, comprising a resin reservoir group.30. A peptide synthesis vessel system according to claim 29 wherein saidresin reservoir group comprises between one and twelve reservoirs.
 31. Apeptide synthesis vessel system according to claim 14, comprising anamino acid reservoir in fluid communication with said second passagewayfor depositing the desired amino acid in said cell.
 32. A peptidesynthesis vessel system according to claim 31 comprising an amino acidreservoir group.
 33. A peptide synthesis vessel system according toclaim 32 wherein said amino acid reservoir group comprises between oneand twenty reservoirs.
 34. A peptide synthesis vessel system accordingto claim 14, comprising a peptide reservoir in fluid communication withsaid second passageway for depositing the completed peptide in saidpeptide reservoir.
 35. A peptide synthesis vessel system according toclaim 34, comprising a peptide reservoir group.
 36. A peptide synthesisvessel system according to claim 35 wherein said peptide reservoir groupcomprises between one and twelve reservoirs.
 37. A peptide synthesisvessel system according to claim 14, comprising a liquid waste containerin fluid communication with said second passageway for depositingsolvent waste therein.
 38. A peptide synthesis vessel system accordingto claim 14, comprising a resin waste container in fluid communicationwith said first passageway for depositing resin waste therein.
 39. Apeptide synthesis vessel system according to claim 14, comprising anamino acid reservoir group and a peptide reservoir group, each of whichis in fluid communication with said second passageway and with one ormore valves for controlling fluid communication between said amino acidand peptide reservoir groups and said cell.
 40. A peptide synthesisvessel system according to claim 14, comprising a dedicated cleavingsolution reservoir and with a dedicated passageway in fluidcommunication with said cleaving solution reservoir and said cell.
 41. Apeptide synthesis vessel system according to claim 18 comprising avalving mechanism in fluid communication with said second, said third,and said fourth passageways that defines a flow path selected from thegroup consisting of sample loops and fluid bypass circuits.
 42. Apeptide synthesis vessel system according to claim 41 further comprisingliquid sensors for determining liquid volume in said sample loop andsaid cell.
 43. A peptide synthesis vessel system according to claim 14,comprising between one and twenty sources for common amino acids or acidderivatives.
 44. A peptide synthesis vessel system according to claim 14comprising a microwave source and a cavity for said vessel with saidsource being in wave communication with said cavity.
 45. A peptidesynthesis instrument according to claim 44, further comprising awaveguide for wave communication between said microwave source and saidcavity.
 46. A peptide synthesis vessel system according to claim 14comprising an infrared temperature sensor capable of measuring andpositioned to measure the infrared radiation emitted by the contents ofsaid cell in said cavity without contacting the contents of said cell.47. A peptide synthesis vessel system according to claim 46 wherein saidtemperature sensor comprises an infrared photosensor that specificallymeasures the temperature of the contents of said cell.
 48. A peptidesynthesis vessel system according to claim 14, further comprising meansfor simultaneously cooling said cell during the application of microwaveradiation.
 49. A peptide synthesis vessel system according to claim 48,further comprising means for simultaneously cooling said cell bycirculating air upon said reaction cell.
 50. A peptide synthesis vesselsystem according to claim 14, wherein said cooling means operatesbetween applications of microwave radiation for at least thedeprotecting, activating, and coupling steps.
 51. A process foraccelerating the solid phase synthesis of peptides, and comprising:deprotecting a protected first amino acid linked to a solid phase resinby admixing the protected linked acid with a deprotecting solution in amicrowave transparent vessel while irradiating the admixed acid andsolution with microwaves; activating a second amino acid by adding thesecond acid and an activating solution to the same vessel whileirradiating the vessel with microwaves; coupling the second amino acidto the first acid while irradiating the composition in the same vesselwith microwaves; and cleaving the linked peptide from the solid phaseresin by admixing the linked peptide with a cleaving composition in thesame vessel while irradiating the composition with microwaves.
 52. Apeptide synthesis process according to claim 51 comprising cooling thevessel during any one or more of the deprotecting, activating, couplingand cleaving steps to prevent heat accumulation from the microwaveenergy from degrading the solid phase support or the peptide.
 53. Apeptide synthesis process according to claim 51, comprising cyclicallyrepeating the steps of deprotecting, activating, and coupling for threeor more amino acids in succession to thereby synthesize a desiredpeptide.
 54. A peptide synthesis process according to claim 51,comprising carrying out said successive deprotecting, activating,coupling, and cleaving steps in the single reaction vessel withoutremoving the peptide from the solid phase resin or from the vesselbetween cycles.
 55. A peptide synthesis process according to claim 51further comprising agitating the admixture with nitrogen gas during oneor more of the deprotecting, activating, coupling, and cleaving steps.56. A peptide synthesis process according to claim 51 comprisingdeprotecting the alpha-amino group of the amino acid.
 57. A peptidesynthesis process according to claim 56 comprising deprotecting thealpha-amino group of the amino acid with a composition suitable forremoving protective chemicals selected from the group consisting ofNÎ±-9-fluorenylmethyloxycarbonyl (Fmoc) and NÎ±-t-butoxycarbonyl (Boc).58. A peptide synthesis process according to claim 51 comprisingdeprotecting the side chain of the amino acid.
 59. A peptide synthesisprocess according to claim 58 comprising deprotecting the side chain ofthe amino acid with a composition suitable for removing t-butyl-basedside chain protecting groups.
 60. A peptide synthesis process accordingto claim 51 further comprising activating and coupling the second aminoacid in situ using a carbodiimide-type coupling reagent.
 61. A peptidesynthesis process according to claim 51 further comprising a cleavingcomposition of trifluoroacetic acid and containing a plurality ofscavenging agents to quench the reactive carbonium ions that originatefrom the protective groups and linkers.